The long-term goal of the research in this proposal is to gain a molecular understanding of chromatin regulation by histone modification with the small ubiquitin-like modifier (SUMO) protein. The post-translational modification (PTM) of histone proteins by a range of chemical groups is observed in all eukaryotes. An extensive body of work has established that the dynamic regulation of histone PTMs and the biochemical relationships between specific PTMs underlie critical processes such as DNA transcription, repair, and replication. One dramatic PTM, the conjugation of histone lysine side-chains with the protein SUMO (termed SUMOylation) occurs widely, from yeast to humans, and is implicated in transcriptional silencing and DNA double-strand break repair. The dysregulation of these critical processes by environmental or genetic factors is linked to many human diseases such as cancers of the blood, brain, breast, and kidneys, to name a few. Therefore elucidating the molecular mechanisms by which histone SUMOylation regulates transcription and gene repair are essential first steps toward devising rational therapeutic strategies for acute human diseases. The biophysical and biochemical characterization of SUMOylated chromatin has until recently been limited by the inability to obtain sufficient quantities of homogeneously SUMOylated histones for in vitro studies, either from cultured cells or by enzymatic means. Hence, essentially nothing is known about the direct and/or indirect mechanisms by which histone SUMOylation influences the structure and function of human chromatin. In order to address this significant gap in our knowledge, we aim to combine the tools of synthetic organic chemistry, biochemistry, molecular and cell biology. Our chemical biology-based approach involves synthesizing site-specifically SUMOylated histones for biochemical and biophysical studies, as well as generating antibodies to investigate the genome-wide occurrence of SUMOylated histones. By adopting methods to study histone SUMOylation both in vitro and in cells, we will gain a comprehensive understanding of the mechanistic roles for this modification in normal growth and in disease progression. The specific research objectives of this proposal are: (1) To elucidate the direct effects of SUMOylation on chromatin structure and stability. (2) To elucidate the biochemical relationship between histone SUMOylation and gene-activating histone modifications, and (3) To investigate the histone SUMOylation state of active and silent regions of human chromatin. The successful completion of these aims will lead to a molecular understanding of SUMO-mediated changes in chromatin structure and function. Identifying new protein- protein interactions in regulatory pathways involving SUMOylated histones may provide new targets for therapeutic invention in diseases arising from the dysregulation of histone modifications. Finally, the methodologies described in this proposal are broadly applicable and will serve as transformative tools for studying SUMO-mediated processes in the context of other key signaling proteins, such as transcription factors, DNA- and histone-modifying enzymes.